Alfredo de Bustos

Cytogenetic diversity of SSR motifs within and between Hordeum species carrying the H genome: H. vulgare L. and H. bulbosum L.

Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)—(AG)n, (AAG)n, (ACT)n and (ATC)n—in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.

CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF THE GENES CODING FOR HIGH MOLECULAR WEIGHT GLUTENIN SUBUNITS IN THREE DIPLOID SPECIES OF AEGILOPS

This work reports the molecular characterization of high molecular weight (HMW) glutenin subunits belonging to the diploid species Aegilops comosa, Aegilops uniaristata, and Aegilops speltoides, which represent three important genomes of the genus Aegilops: M, Un, and S, respectively. Besides improving our knowledge of Aegilops glutenins, which is useful in wheat breeding programs, this study examines the phylogenetic relationships between the glutenin genes of an important group of species of the tribe Triticeae. The new glutenin proteins examined (coded by Glu‐Mx, Glu‐My, Glu‐Unx, Glu‐Uny, and Glu‐Sy) showed the same general characteristics as those previously described in other species of the tribe Triticeae. A phylogenetic study of the relationships between the new members of the Glu gene family and other known sequences of glutenin genes of the genera Aegilops, Triticum, and Secale was performed. In this analysis, the x‐ and y‐type glutenin subunits showed similar interspecific relationships and differentiated rye from wheat and the Aegilops species. In both cases, the glutenin genes of Aegilops tauschii appeared closely related to the glutenins of the D genome, which, in turn, are closely related to those of Aegilops umbellulata. The homologous gene subunits of A. comosa and A. uniaristata from the section Comopyrum were grouped together. The glutenin gene of A. speltoides was grouped with the wheat B genome genes. This result reinforces the close relationships between the S and B genomes of Aegilops and Triticum, respectively.

The evolutionary history of sea barley (Hordeum marinum) revealed by comparative physical mapping of repetitive DNA.

BACKGROUND AND AIMS Hordeum marinum is a species complex that includes the diploid subspecies marinum and both diploid and tetraploid forms of gussoneanum. Their relationships, the rank of the taxa and the origin of the polyploid forms remain points of debate. The present work reports a comparative karyotype analysis of six H. marinum accessions representing all taxa and cytotypes. METHODS Karyotypes were determined by analysing the chromosomal distribution of several tandemly repeated sequences, including the Triticeae cloned probes pTa71, pTa794, pAs1 and pSc119·2 and the simple sequence repeats (SSRs) (AG)10, (AAC)5, (AAG)5, (ACT)5 and (ATC)5. KEY RESULTS The identification of each chromosome pair in all subspecies and cytotypes is reported for the first time. Homologous relationships are also established. Wide karyotypic differences were detected within marinum accessions. Specific chromosomal markers characterized and differentiated the genomes of marinum and diploid gussoneanum. Two subgenomes were detected in the tetraploids. One of these had the same chromosome complement as diploid gussoneanum; the second subgenome, although similar to the chromosome complement of diploid H. marinum sensu lato, appeared to have no counterpart in the marinum accessions analysed here. CONCLUSIONS The tetraploid forms of gussoneanum appear to have come about through a cross between a diploid gussoneanum progenitor and a second, related-but unidentified-diploid ancestor. The results reveal the genome structure of the different H. marinum taxa and demonstrate the allopolyploid origin of the tetraploid forms of gussoneanum.

On the allopolyploid origin and genome structure of the closely related species Hordeum secalinum and Hordeum capense inferred by molecular karyotyping

Background and Aims To provide additional information to the many phylogenetic analyses conducted within Hordeum , here the origin and interspecific affinities of the allotetraploids Hordeum secalinum and Hordeum capense were analysed by molecular karyotyping. Methods Karyotypes were determined using genomic in situ hybridization (GISH) to distinguish the sub-genomes and , plus fluorescence in situ hybridization (FISH)/non-denaturing (ND)-FISH to determine the distribution of ten tandem repetitive DNA sequences and thus provide chromosome markers. Key Results Each chromosome pair in the six accessions analysed was identified, allowing the establishment of homologous and putative homeologous relationships. The low-level polymorphism observed among the H. secalinum accessions contrasted with the divergence recorded for the sub-genome of the H. capense accessions. Although accession H335 carries an intergenomic translocation, its chromosome structure was indistinguishable from that of H. secalinum . Conclusion Hordeum secalinum and H. capense accession H335 share a hybrid origin involving Hordeum marinum subsp. gussoneanum as the genome donor and an unidentified genome progenitor. Hordeum capense accession BCC2062 either diverged, with remodelling of the sub-genome, or its genome was donated by a now extinct ancestor. A scheme of probable evolution shows the intricate pattern of relationships among the Hordeum species carrying the genome (including all H. marinum taxa and the hexaploid Hordeum brachyantherum ).

CHARACTERIZATION OF THE NBS1 GENE AND ANALYSIS OF THE EXPRESSION OF HOMOLOGOUS AND HOMOEOLOGOUS MRN COMPLEX GENES IN MEIOCYTES AND SOMATIC CELLS OF DIFFERENT WHEAT SPECIES

The products of the Nbs1, Mre11, and Rad50 genes form the MRN complex. This complex is involved mainly in DNA repair, but it also has roles in meiosis, replication, telomere maintenance, and cell cycle checkpoint signaling. Its study is therefore of great importance if our knowledge of these important cell processes is to improve. To date, few studies have examined the MRN complex in plants. The present work describes the molecular characteristics of the Nbs1 gene in diploid and polyploid species of wheat. The expression of MRN complex homologous genes in meiocytes and in somatic cells treated with genotoxic agents is also examined. In addition, the expression of MRN complex homoeologous genes is analyzed in polyploid wheat species to determine the function of the duplicated genes. The characterization of Nbs1 in wheat species revealed its low sequence homology with the Nbs1 gene of other organisms. However, the homologous Nbs1 genes of wheat showed very great similarity. All the Nbs1 wheat genes examined had the characteristic domains necessary for proper protein function. A putative second BRCT domain and a new SQ motif are here described for the first time in plants. Higher rates of Nbs1 expression were detected in meoicytes. In leaf and root cells, basal expression was observed even after treatments with genotoxic agents. Overall, Nbs1 was the MRN complex gene most weakly expressed. MRN complex homoeologous genes were expressed at different levels in polyploid species; indeed, expression was biased in some cases. No silencing of copies was detected in any of the samples analyzed, indicating maintenance of the function of MRN complex homoeologous genes.

Sequencing of long stretches of repetitive DNA

Repetitive DNA is widespread in eukaryotic genomes, in some cases making up more than 80% of the total. SSRs are a type of repetitive DNA formed by short motifs repeated in tandem arrays. In some species, SSRs may be organized into long stretches, usually associated with the constitutive heterochromatin. Variation in repeats can alter the expression of genes, and changes in the number of repeats have been linked to certain human diseases. Unfortunately, the molecular characterization of these repeats has been hampered by technical limitations related to cloning and sequencing. Indeed, most sequenced genomes contain gaps owing to repetitive DNA-related assembly difficulties. This paper reports an alternative method for sequencing of long stretches of repetitive DNA based on the combined use of 1) a linear vector to stabilize the cloning process, and 2) the use of exonuclease III for obtaining progressive deletions of SSR-rich fragments. This strategy allowed the sequencing of a fragment containing a stretch of 6.2 kb of continuous SSRs. To demonstrate that this procedure can sequence other kinds of repetitive DNA, it was used to examine a 4.5 kb fragment containing a cluster of 15 repeats of the 5S rRNA gene of barley.

A novel FISH technique for labeling the chromosomes of dinoflagellates in suspension

Dinoflagellates possess some of the largest known genomes. However, the study of their chromosomes is complicated by their similar size and their inability to be distinguished by traditional banding techniques. Dinoflagellate chromosomes lack nucleosomes and are present in a liquid crystalline state. In addition, approaches such as fluorescent in situ hybridization (FISH) are problematic because chromosomes are difficult to isolate from the nuclear membrane, which in dinoflagellates remains intact, also during mitosis. Here we describe a novel, reliable and effective technique to study dinoflagellate chromosomes by physical mapping of repetitive DNA sequences in chromosomes in suspension (FISH-IS), rather than on a microscope slide. A suspension of non-fixed chromosomes was achieved by lysing the cells and destabilizing the nuclear envelope. This treatment resulted in the release of the permanently condensed chromosomes in a high-quality chromosomal suspension. Nevertheless, slide preparations of the chromosomes were not suitable for conventional FISH because the nuclear integrity and chromosomal morphology was destroyed. Our newly developed, simple and efficient FISH-IS technique employs fluorescently labeled, synthetic short sequence repeats that are hybridized with suspended, acetic-acid-pretreated chromosomes for 1 h at room temperature. The method can be successfully used to discriminate single chromosomes or specific chromosomal regions, depending on the specificity of the repeat sequences used as probes. The combination of FISH-IS and flow sorting will improve genomic studies of dinoflagellates, overcoming the difficulties posed by their huge genomes, including long stretches of non-coding sequences in multiple copies and the presence of high-copy-number tandem gene arrays.

Comparative analysis of gene expression among species of different ploidy

Interspecific comparative studies require that expression data be comparable among species, and when species with different levels of ploidy are contemplated the relative expression per cell should be obtained for accurate comparisons to be made. Quantitative reverse-transcription-PCR is the most popular and sensitive technique for the detection and quantification of mRNA in gene expression analysis. In recent years it has become clear that the choice of reference genes for the normalization of expression data is very important. Several studies have shown that the expression of the traditional housekeeping genes varies under certain situations; their use as reference genes in quantitative PCR assays can therefore lead to errors when interpreting the relative expression of target genes. Normalizing with respect to endogenous genes showing a constant level of expression per cell across species, however, provides an easy way of obtaining comparable expression data for other genes in those species. In this work, the validity of several candidate genes was examined across four diploid and polyploid species of the genera Triticum and Aegilops. Candidate reference genes were chosen among the traditional housekeeping genes used in quantitative PCR analysis, as well as others found to have stable levels of expression under different conditions in other studies. After the analyses, candidate genes were gathered into two groups according to the different levels of expression per cell seen in polyploid species. For the four species studied, two genes suitable for normalization procedures in interspecific studies were identified: cell division control protein and malate dehydrogenase. Both showed a constant number of transcripts per cell, independent of the level of ploidy.